Fig 2: a–e Confocal microscopy images of SGN nuclei co-stained for ?H2AX in combination with fibrillarin (a, b), coilin (c), histone H4K20me3 (d) and HP1? (e). They illustrate the direct association of PDDF with a triad of structures: the nucleolus (a, c, e), Cajal bodies, immunolabeled for fibrillarin (b) or coilin (c), and perinucleolar heterochromatin clumps immunolabled for the tri-methylated histone H4K20me3 (d) or HP1? (e). f Double immunolabeling for 53BP1 and TMG-cap illustrate the distribution of the nuclear speckles of splicing factors labelled with the anti-TMG-cap antibody and the close association of a PDDF with a speckle in the vicinity of the nucleolus. 45d post-IR. Scale bar = 5 µm

Fig 3: Mouse interdigital cells at day 13.5 pc immunolabeled for ?H2AX (A–C) in combination with MDC1 (A´), RAD50 (B´) and 53BP1 (C´) to show the activation of DDR mediators prior to overt cell degeneration. A´´, B´´, and C´´ are merged images. (D) Pixel intensity plot of the region indicated by red arrow showing the labeling distribution of 53BP1 (red) and ?H2AX (green) in an interdigital cell similar to that shown in (C) Note the presence of isolated ?H2AX foci and a region of colocalization with 53BP1. (E) Chick interdigital cells at id 6 immunolabeled for ?H2AX (E) and pATM (E´). E´´ is the merged image. Inset is a detailed view of the cell indicated by asterisk. Bars in (A–C) = 10 µm; bar in (E) = 30 µm.

Fig 4: Brca1 and 53BP1 Together Promote Resection-Dependent Slow DSB Repair in G1(A) ?H2AX foci in G1 82-6 and CJ179 cells treated with siBrca1. Data are mean ± SEM.(B) pRPA foci in G1 HeLa cells treated with si53BP1 and/or siBrca1. Data are mean ± SEM.(C) End joining events in GC92 cells treated with si53BP1 and/or siBrca1. Data are mean ± SEM.(D) ?H2AX foci in G1 CJ179 and end joining events in GC92 cells. Cells were treated with siBrca1 and transfected with GFP, RFP or FLAG-Brca1-WT constructs, and GFP+, RFP+ or FLAG+ cells were analyzed. Data are mean ± SEM.(E) ?H2AX foci in G1 Brca1-WT and Brca1-?BRCT MEFs treated with siArtemis. Data are mean ± SEM.(F) pRPA foci in G1 Brca1-WT and Brca1-?BRCT MEFs. Data are mean ± SEM.See also Figure S5.

Fig 5: Bulky DNA adducts induce RAD51 foci formation in absence of replication fork stalling or -collapse.a Schematic of DNA repair and damage tolerance pathways induced by low doses of BPDE, which forms bulky adducts on guanine. NER: nucleotide excision repair; TLS: translesion synthesis; HR: homologous recombination. b Co-localization of ?H2AX (green) and replication forks (CldU, red) in U2OS cells after 1 h release from 1650 nM BPDE treatment for 20 min. Images are representative of n = 2. Scale bars: 10 µm. c Percentages of U2OS cells with >8 ?H2AX foci after release from increasing concentrations of BPDE. n = 3 d BPDE-induced RAD51 foci. Images are representative of n = 3. Scale bars: 10 µm. e Percentages of U2OS cells with >5 RAD51 foci after release from increasing concentrations of BPDE. n = 3. f Percentages of stalled forks after release from BPDE. U2OS cells were pulse-labeled with CldU, treated with BPDE during the CldU pulse, and released into IdU. n = 4 (1650 nM n = 3). g Percentages of cells with >10 53BP1 foci after release from increasing concentrations of BPDE. n = 3. h Top: Protein levels of RAD51 and a-Tubulin (loading control) in U2OS cells after 48 h depletion with RAD51 or non-targeting (nonT) siRNA. Images are representative of n = 2. Bottom: Strategy for DNA fiber labeling. i Percentages of stalled forks after release from 50 nM BPDE as shown in (h) in cells treated with nonT or RAD51 siRNA for 48 h. n = 3. j Percentages of control- or RAD51-depleted U2OS cells with >10 53BP1 foci after release from 50 nM BPDE. n = 3. Source data are provided as a Source Data file. The means and SEM (bars) of at least three independent experiments are shown. Asterisks indicate p-values compared to solvent unless indicated otherwise (one-way ANOVA and Dunnett’s test for C, E, F, G; one-sided student’s t-test for I, J, *p < 0.05, **p < 0.01, ***p < 0.001).